Tipifarnib Reveals the Need for Precision Drugging in HNSCC

RAS proteins are small GTPases activated upon GTP binding to stimulate downstream RAF/MEK/MAPK signaling for cell growth. As membrane localization of RAS proteins is required for their activation, pharmacologic inhibition of RAS post-translational modifications, such as C-terminal farnesylation (by inhibiting the enzyme farnesyl transferase that transfers farnesyl group to the C-terminal of RAS), could potentially inactivate RAS. Till now, four farnesyl transferase inhibitors (FTIs), namely L-778,123, BMS-214662, tipifarnib, and lonafarnib have been clinically evaluated in various cancers, including HNSCC (Table ​(Table2).2). Their effects in HNSCC patients are discussed below.

Table 2

Failure of early non-precision clinical trials for RAS inhibitors or farnesyl transferase inhibitors (FTIs) involving HNSCC patients.

Clinical trial ID	Clinical trial (tumor type; N)	Target(s)	Drug(s)	No. of HNSCC patients	HNSCC patient outcome	Outcomes of non-HNSCC patients	Ref
N.A.	Phase I (HNSCC and NSCLC; N=9)	FT/GGTase-I	L-778,123 [with standard radiotherapy]	3	2 had complete clinical respones with no evidence of disease at follow-up examinations, but MTD of th L-778,123/radiation combination was not well-defined.	Of the 4 evaluable NSCLC patients, 3 had a complete response and 1 had a partial response.	41
NCT00003430	Phase I (HNSCC and unspecified solid tumors; N=N.A.)	FT/GGTase-I	L-778,123	N.A.	“Completed” but results unavailable.	N.A.	N.A.
NCT00006242	Phase I (multiple solid tumors; N=44)	FT	BMS-214662	2	No objective responses reported.	Antitumor activity was observed in 1h I.V. infusion in patients. A pancreatic cancer patient has prolonged stable disease for more than five years under BMS-214662 treatment; A NSCLC patient has 40% size reduction in liver metastasis and complete shrinkage in one of three brain metastases.	42
NCT00005973	Phase I (multiple solid tumors; N=30)	FT	BMS-214662	6	No objective responses reported.	A minor response in a NSCLC patient; A weekly 1h infusion did not provide sustained and substantial enough FTase inhibition for apoptosis.	43
NCT00038584	Phase Ib (HNSCC; N=37)	FT	Lonafarnib	37	“Clinical responses” were seen at all lonafarnib dose levels (oral 100mg twice daily (b.i.d.), 200mg b.i.d., or 300mg b.i.d. for 8 to 14 days before surgical resection), with marked tumor reduction in 4 of 22 evaluable patients. 3 of 17 treated patients demonstrated partial responses.	N.A.	44
NCT00073450	Phase II (HNSCC; N=15)	FT	Lonafarnib	15	7 patients (47%) had stable disease; Well-tolerated but no objective responses observed in the first 15 patients enrolled, and the study is terminated to further accrual.	N.A.	45
NCT00102635	Phase I (HNSCC; N=N.A.)	FT	Lonafarnib [with 4-HPR]	N.A.	Terminated due to slow accrual.	N.A.	N.A.

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L-778,123: a Phase I trial aimed to determine L-778,123’s maximal tolerated dose (MTD) in combination with radiotherapy in 9 advanced cancer patients (3 with stage IV HNSCC, 6 with NSCLC)⁴¹. Among the 3 HNSCC patients, despite one patient falling short of complete treatment due to toxicity, the remaining 2 patients have demonstrated complete responses with no evidence-of-disease (NOD), at follow-up examinations. Ras mutational statuses were not clearly reported. Despite complete responses observed, the MTD of the L-778,123/radiation combination could not be defined⁴¹. It was also noted that another Phase I clinical trial for L-778,123 was conducted by the Memorial Sloan Kettering Cancer Center and National Cancer Institute (NCT00003430) for patients with recurrent or refractory solid tumors including HNSCC, but no trial result was published.

BMS-214662: a potent FTI of the 1,4-benzodiazepine class with IC₅₀ value of low nM range in vitro. Two HNSCC patients involved in a mixed solid tumor Phase I trial, were treated with BMS-214662 and did not show objective responses, while another Phase I BMS-214662 trial with HNSCC patients did not report patient outcomes in detail^42,43.

Lonafarnib (SCH66336): a tricyclic peptidomimetic inhibitor being one of the first FTIs underwent human clinical trials for cancer, including both Phase I and II trials involving HNSCC patients. An early Phase Ib trial result released in a conference proceeding reported clinical responses at all lonafarnib doses tested in patients prior surgery (100 to 300mg twice daily), with marked tumor size reduction in 4/22 evaluable patients⁴⁴. A later Phase II lonafarnib trial in chemo-refractory advanced HNSCC showed that among 15 patients who previously failed platinum-based therapies, 7 had stable diseases in a minimum of 3 cycles of treatment, while 1 patient was stable for 8 cycles of treatment for 220 days total. Further, lonafarnib was well-tolerated with no grade 3 or 4 hematologic toxicities⁴⁵. Among these advanced HNSCC patients, these stable disease cases with lonafarnib suggested potential therapeutic efficacy of FTIs for HNSCC, which was later shown by tipifarnib. Interestingly, lonafarnib was recently approved by the FDA (Nov, 2020) for the treatment of a form of premature aging disease called Hutchinson-Gilford progeria syndrome, and progeroid laminopathies to reduce their risk of death by preventing the build-up of “defective progerin or progerin-like protein” that causes accelerated heart failure.

Tipifarnib: a potent FTI (Supplementary Fig. 2), with IC₅₀ values of 0.86nM and 7.9nM against lamin B and K-RasB peptide substrates in vitro⁴⁶. With the realization that HRAS mutations can cause oncogene addiction and activated MAPK signaling in cancer, tipifarnib trials have sought to investigate its effectiveness in pan-cancer patients with and without HRAS mutations. In a recent phase II trial (NCT02383927), tipifarnib demonstrated a 53% overall response rate (13/23 patients) among HNSCC patients⁴⁷. Furthermore, significant clinical activity was noted in patients with recurrent and metastatic HNSCC harboring HRAS mutation with high variant allele frequency (VAF) of >20%. Eight out of 15 HNSCC patients meeting such a criterion had partial responses, while additional 5 demonstrated stable diseases (53% response rate) with tipifarnib⁴⁷. Tipifarnib also demonstrated modest clinical activity in patients with recurrent, metastatic HRAS-mutant salivary gland cancer, and urothelial carcinoma. Among 12 evaluable HRAS-mutant recurrent metastatic salivary gland cancer (SGC) patients, one demonstrated partial response and 7 had stable disease with median duration of response of 9 months⁴⁸. In this small SGC cohort, the kind of HRAS variants and allele frequency did not correspond to clinical outcome with tipifarnib. Nevertheless, these promising clinical findings provide strong evidences for the use of HRAS mutations in HNSCC as predictive biomarkers for tipifarnib sensitivity. In general, tipifarnib was well-tolerated, with fatigue, myelosuppression, nausea, and vomiting being the most common adverse events (all grades) observed⁴⁷. Currently, the FDA has granted fast-track designation to tipifarnib for HRAS-mutant HNSCC whose disease progressed on platinum therapy⁴⁹. An ongoing international Phase II trial is specifically evaluating tipifarnib efficacy in HNSCC patients with high HRAS-mutant VAF (NCT03719690). It is very likely that HRAS-mutant HNSCC may represent the first precision medicine specific for HNSCC.

Alongside, studies are ongoing to start tackling potential tipifarnib-resistance in HNSCC. Using HRAS-mutant HNSCC patient-derived xenografts (PDXs), tipifarnib-resistance was identified to involve aberrant apoptosis and angiogenesis⁵⁰. Nevertheless, their involvement and clinical relevance in HNSCC remains unknown. A recent CRISPR-screen has already identified potential combination strategy of tipifarnib with autophagy inhibitors to prepare for even more effective tipifarnib treatment for HRAS-mutant HNSCC⁵¹.

Drugging HNSCC with KRAS Germline Variants by Cetuximab Addition

As tipifarnib demonstrates good clinical efficacies in HRAS-mutated HNSCC, salivary gland carcinoma, and urothelial carcinoma^48,52,53, other RAS genes have gained attention for precision medicine development. New RAS inhibitors, AMG-510 and MRTX849, have been recently developed and showed clinical promises in patients with KRAS p.G12C-mutated solid tumors (NCT03600883, NCT04330664)⁵⁴. In fact, AMG-510 has recently been FDA-approved for the treatment of KRAS p.G12C-mutated locally advanced or metastatic NSCLC (May, 2021). Unlike HRAS, the mutation rate of KRAS is relatively low in HNSCC (1/512; 0.2%). Yet, we recently reported that KRAS mutation rate can found in ~3.5% of advanced HNSCC⁵⁵. In metastatic larynx cancer, KRAS mutation may account for ~6% cases⁵⁶. Furthermore, KRAS mutations appear to be a poor prognostic biomarker for advanced HNSCC, with shorter disease-free survival in KRAS-mutant vs. WT patients⁵⁵.

Among all new KRAS inhibitors under clinical evaluation, only AMG-510, MRTX849, and LY3499446 have entered phase II settings⁵⁷. Yet, the low rate of KRAS somatic mutations in HNSCC presents a challenge for clinical assessment of KRAS inhibitors with KRAS-status stratification.

Interestingly, Weidhaas et al. recently performed a secondary analysis of a randomized phase III HNSCC trial and found that patients with an oncogenic germline KRAS variant (a let-7 microRNA-binding site polymorphism in the 3’ untranslated region of KRAS) have significantly better clinical outcomes with cetuximab (EGFR monoclonal antibody) addition to radiotherapy plus cisplatin regimen⁵⁸. Furthermore, these germline KRAS-variant HNSCC patients were found to have increased plasma TGF-β1, potentially contributing to immunosuppression in these patients. Thus, targeting with cetuximab may help these patients overcome TGF-β1–induced immunosuppression. In fact, this very same KRAS germline variant also predicts good clinical response to cetuximab monotherapy in otherwise somatic KRAS-WT metastatic colon cancer patients⁵⁹. Thus, KRAS germline variant guiding combination therapy with cetuximab, and potentially radiotherapy/cisplatin regimen should be further evaluated in larger prospective trial in HNSCC. The main side effects reported in the trial were skin reaction, some with grade 3 to 4 mucositis⁵⁸.

Exceptional Erlotinib responses for MAPK1-mutant HNSCC

About 5% of HNSCC patients harbor somatic mutations of MAPK1 (ERK2) gene¹². In 2015, Van Allen and Lui et al. reported the first HNSCC exceptional responder (with Stage III advanced oral cancer) who exhibited a complete response (>30 months) to a 13-day erlotinib treatment⁶⁰. This clinical finding is highly unexpected given another EGFR targeting agent, cetuximab often demonstrates moderate activities in HNSCC patients in general. Whole-exome characterization of pre-treatment biopsy showed no EGFR aberrations, but the presence of a somatic MAPK1 p.E322K mutation using an EGFR-network bioinformatics approach. This mutation was then functionally characterized to be a potent driver for constitutive ERK activation and HNSCC cell growth¹². Subsequent investigation revealed its ability to drive robust EGFR hyperactivation by enhancing autocrine amphiregulin release from HNSCC cells, thus hyperactivating EGFR signaling⁶¹, and rendering hypersensitivity to an EGFR kinase inhibitor, erlotinib. The study was later extended to additional MAPK1 mutations in HNSCC, and led to the finding that MAPK1 p.D321N (also hyperactivates MAPK and p-EGFR) could also confer heightened sensitivity to erlotinib in HNSCC in vivo, while MAPK1 p.R135K mutation (moderately activating p-EGFR) conferred moderate level of erlotinib sensitivity¹². Importantly, both MAPK1 p.D321N and p.R135K mutations exist in recurrent HNSCC in Asia¹². These results suggested that MAPK (or ERK) activities could be associated with erlotinib responses in HNSCC. In fact, results from the randomized clinical trial (NCT00779389) involving the first HNSCC exceptional responder eventually concluded that baseline tumoral p-MAPK (p-ERK) levels were inversely correlated with tumor size post-erlotinib treatment, showing that MAPK activation status is likely an indicator of EGFR-addiction in HNSCC, and thus predictive of clinical responses to erlotinib in HNSCC patients⁶². Overall, the clinical trial reported brief exposure to erlotinib was well-tolerated in HNSCC, with acneiform rash and diarrhea being major side effects that are commonly observed with EGFR inhibitors⁶¹.

Interestingly, based on current genomic findings, US TCGA-HNSCC patients with primary tumors universally harbor MAPK1 p.E322K mutations, while an Asian primary/recurrent HNSCC cohort showed a wide spectrum of p.E322K/D321N/R135 mutations¹². As EGFR kinase inhibitors (e.g., erlotinib, gefitinib) are known to be clinically safe for treatment of lung cancer, these clinical and findings provide important scientific evidences supportive of precision trials for MAPK1-mutated, yet EGFR-addicted HNSCC.

BRAF p.V600E-mutated Ameloblastoma are Exceptionally Sensitive to BRAF/MEK Inhibitors

Ameloblastoma is a rare head and neck tumor (1.79/10,000,000 persons/year) found in the jaw area near the molar⁶³. Though rare, high rates of BRAF p.V600E hotspot mutation (33.3–82%) have enticed growing interest for BRAF-targeting, as ameloblastoma often requires extensive facial surgeries, compromising quality-of-life, yet, frequently recurs^64–68.

Five exceptional responder reports have been documented with dabrafenib or vemurafenib monotherapies, and dabrafenib/trametinib combination (Table ​(Table3).3). Kaye et al. first reported an African American ameloblastoma patient with recurrent metastases bearing somatic BRAF p.V600E mutation (detected by mutant-specific antibody with immunohistochemistry), treated with compassionate dabrafenib (150mg twice daily) plus trametinib (2mg once daily) and exhibited immediate marked tumor reduction at day 4, followed by disappearance of lung metastases and shrinkage of head and neck lesions at week 8, and persistent antitumor responses even at 20 weeks⁶⁹. This combination was well-tolerated clinically. Another remarkable clinical responses to this combination was also observed in a 26-year old recurrent metastatic ameloblastoma patient showing complete dissolution of lung metastasis and primary tumor at 30 weeks after treatment (NCT02534649)⁷⁰. The NCT02367859 trial also reported a stable disease for a BRAF p.V600E-mutated ameloblastoma patient. These cumulative responder cases strongly suggest the potential therapeutic benefits of BRAF/MEK inhibition in BRAF p.V600E-mutated ameloblastoma (note: such combination is FDA-approved for BRAF p.V600E-mutated thyroid cancer and melanoma). Besides, two other recurrent BRAF p.V600E-mutated ameloblastoma patients have also responded dramatically to dabrafenib monotherapy. These include an 83-year-old patient treated with low dose dabrafenib (75mg twice daily) showing 75% tumor size reduction at 12 months⁷¹, and another recurrent patient responding to dabrafenib with >90% tumor shrinkage⁷². Lastly, besides dabrafenib, the use of vemurafenib has also caused persistent and marked tumor shrinkage (>60% for 11 months) in a recurrent BRAF p.V600E-mutated ameloblastoma patient⁷³. Common reported side effects for these BRAFi, MEKi and their combinations include asthenia, rash, arthralgia, nausea, pyrexia, fatigue and headache, etc., and they, in general, showed good toxicity profiles in patients⁷⁰. Thus, BRAF p.V600E-mutated ameloblastoma could be pharmacologically vulnerable for precision BRAF-targeting.

Table 3

Exceptional responders to BRAF and/or MEK inhibitors among patients with BRAF p.V600E-mutated recurrent/metastatic ameloblastoma.

Cancer type	Tumor stage	Target(s)	Drug(s)	Response by RECIST and PERCIST criteria	Duration of treatment	Details on tumor volume reduction	Ref
BRAFp.V600E Ameloblastoma	Metastatic	MEK1/2 and BRAF	Dabrafenib and Trametinib	Partial response	20 weeks	Visible tumor size reduction shown in pre- and post-treatment PET-CT images 8 weeks after treatment onset (reduction percentage unreported).	69
	Metastatic	MEK1/2 and BRAF	Dabrafenib and Trametinib	Complete response	30 weeks	Complete response ongoing 30 weeks after onset of treatment.	70
	Recurrent	BRAF	Dabrafenib	Partial response	12 months	75% tumor size reduction at 8 months with 50% standard dose of dabrafenib for metastatic melanoma, and durable response continued at 12 months.	71
	Recurrent	BRAF	Dabrafenib	Partial response	73 days	No change in tumor size but over 90% tumor volume reduction characterized by degeneration and squamous differentiation of the inner parts of tumors.	72
	Recurrent	BRAF	Vemurafenib	Partial response	11 months	Reduction of lesion size from 24×21×19mm to 18×13×14mm 11 months after treatment initiation.	73

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Remarkable clinical responses to MAPK inhibitors in BRAF-mutated HNSCC

Unlike ameloblastoma, BRAF mutations only occur in ~1.8% of HNSCC, and the clinical responsiveness of BRAF-mutated HNSCC patients to BRAF inhibition remains under-explored. Yet, a recent Phase I trial (NCT01781429) did report a BRAF p.G469A-mutated head and neck cancer patient with partial response to ulixertinib (an ERK1/2 inhibitor) per RECIST criteria (> 30% tumor size reduction)⁷⁴. Though such a dramatic ulixertinib response only lasted for 4.9 months in this patient, a little fall short of the NCI’s criteria for exceptional responders (6-months), this report does provide clinical evidences supportive of future ulixertinib trials in relation to BRAF-mutational status for this relatively safe drug. In the trial, the most common treatment-related adverse effects were rash, diarrhea, fatigue, and nausea, with no grade 4 or 5 treatment-related adverse effects reported.

MAPK mutations modulate ErbB3 activation in HNSCC: potential therapeutic consideration

ERBB3 is a new target for HNSCC. Its activation level (p-ErbB3) is significantly associated with poorer HNSCC patient survival⁷. Various ErbB3 inhibitors are under development. Among which, CDX-3379, an anti-ErbB3 monoclonal antibody, has demonstrated antitumor activity resulting in tumor shrinkage in 42% of HNSCC patients, with grade 1 to 2 diarrhea, fatigue, and acneiform dermatitis being the most often treatment-related toxicities⁷⁵.

A recent study showed that multiple MAPK pathway mutations found in HNSCC patient tumors, including BRAF p.V600E, HRAS p.G12V, MEK1 p.K57N, MEK2 p.F57L, MAPK1 p.E322K, MAPK1 p.D321N, ARAF p.S214F, ARAF p.P508L, as well as wildtype A/BRAF genes could significantly suppress p-ErbB3 levels in HNSCC cells⁷. Moreover, pharmacologic inhibition of MAPK1/3 activity by GDC-0994 has resulted in p-ErbB3 activation in MAPK-mutant HNSCC primary cultures and cell models, but not in WT counterparts, indicating that MAPK activating mutations can modulate p-ErbB3 levels in HNSCC. This was further supported by findings in HNSCC patient tumors with high allele frequencies of HRAS p.G12S and MAPK1 p.D321N mutations (>30-40% allele frequency) expressing low levels of p-ErbB3. Thus, MAPK mutational status or ERK activities can modulate ErbB3 activation level in HNSCC, suggesting the need for precautions when using ErbB3 inhibitors in MAPK-mutant HNSCC patients. Further clinical studies should be conducted to determine if perhaps, ErbB3 inhibitors may/should be avoided in MAPK-mutant HNSCC patients.

MAPK-mutant HNSCC are CD8⁺T cell-inflamed: implications for T cell-based immunotherapies

HPV(+)HNSCC patients are known to respond well to T cell-based immunotherapies, including PD1 inhibitors pembrolizumab and nivolumab^76–79. This is at least partially contributed by elevated immune infiltrates in HPV(+)HNSCC tumors^76–82. Numerous PD1 inhibitor trials confirmed that HPV(+)HNSCC patients have reduced hazard ratios for death (HR=0.82, p=0.0316) when compared to standard treatment⁸³. For recurrent and metastatic HNSCC, HPV(+)HNSCC patients demonstrated a better objective response rate to durvalumab than HPV(-)HNSCC patients (29.4% vs 10.8%, with median OS=10.2 months 5.0 months)⁸⁴. However, HPV status was not associated with clinical responses to nivolumab in the CheckMate 141 study⁸⁵. Recent bioinformatic analysis of HNSCC patient tumors showed that MAPK-mutant HNSCC tumors, irrespective of HPV status, also have elevated CD8⁺T cell and dendritic cell infiltrations, together with immune-active cytolytic and interferon-gamma signatures in situ, suggestive of an active cytolytic CD8⁺T-inflamed status in these tumors. Interestingly, this CD8⁺T-inflamed and cytolytic feature was not shared by HNSCC tumors bearing PI3K, NOTCH, JAK/STAT, WNT, NF-κB, or TGFβ/Smad pathway mutations⁷. The study further demonstrated that ectopic expression of MAPK pathway mutations (mouse HRAS p.G12V, mouse MAPK1 p.D319N and p.E320K), but not respective WT-counterparts, could directly shape the HNSCC immune tumor microenvironment by attracting CD8⁺T cell infiltration in immunocompetent mouse allografts⁷. Whether such MAPK mutation-driven CD8⁺T cell infiltration was caused by activated MAPK signaling or the associated neoantigens warrants further investigations. It also remains to be determined if these CD8⁺T cells are clonal or not. Using TCGA-HNSCC dataset, Lyn et al. also showed immune-enhancing signatures associated with HRAS mutation, including elevated CD8⁺ markers and HLA gene expressions, high cytolytic activity, and immune scores⁸⁶.

Since CD8⁺T cells are effective antitumor immune cells, its markedly increased level in MAPK-mutant HNSCC tumors may tie to PD1 inhibitor response. In fact, subsequent retrospective analysis of anti-PD1 immunotherapy cohorts did reveal that MAPK pathway mutations could also predict anti-PD1 immunotherapy outcome in advanced and metastatic oral cancer patients⁷, with 2-3 times longer median survival than WT patients⁷. Thus, such a highly immunoactivated status of MAPK-mutant HNSCC tumors may potentially outweigh the oncogenic effects of these activating mutations, and constitute better clinical outcome to ICI in HNSCC patients. Large prospective ICI clinical investigation with known MAPK-mutation status should be warranted to determine the clinical efficacy of ICI therapy in HNSCC patients for precision immunotherapy.